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Freshly isolated viable rat hepatocytes were separated into five subpopulations on shallow discontinuous Percoll density gradients. The periportal marker enzymes alanine aminotransferase (ALT), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) showed gradients of increasing activity from the subpopulation of least density (band 1, rho = 1.07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamate dehydrogenase (GDH) showed gradients of decreasing activity from band-1 cells to band-5 cells. Glutamine synthetase (GS), which is confined to the two or three cell layers around the hepatic venule, was almost entirely restricted to band-1 hepatocytes. Band-5: band-1 ratios of enzyme activity were as follows: ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions for up to 72 h, whereas the ratios for MDH and GDH decreased and increased respectively towards unity. Band-1 hepatocytes exhibited greater cytotoxicity than band-5 cells after incubation with carbon tetrachloride or paracetamol. These perivenous-selective toxins produced greater decreases in cell viability and greater release of ALT and LDH from band-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepatocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal-selective toxins). It is concluded that band-5 hepatocytes are enriched in periportal cells, whereas band-1 hepatocytes are enriched in perivenous cells. Isolation of hepatocyte subpopulations by Percoll density-gradient centrifugation has the considerable advantage that periportal and perivenous cells can be obtained from the same liver.

Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations